Anti-gm-csf antibodies

ABSTRACT

The present invention provides recombinant antigen-binding regions, antibodies and functional fragments thereof that are specific for GM-CSF, which plays an integral role in various disorders or conditions. These antibodies, accordingly, can be used to treat, for example, inflammatory diseases such as rheumatoid arthritis. Antibodies of the invention also can be used in the diagnostics field, as well as for further investigating the role of GM-CSF in the progression of various disorders. The invention also provides nucleic acid sequences encoding the foregoing antibodies, vectors containing the same, pharmaceutical compositions and kits with instructions for use.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. application Ser. No.12/923,363, filed Sep. 16, 2010, which is a Divisional of U.S.application Ser. No. 11/914,599, which is the US National Stageapplication of PCT/EP2006/004696, filed May 17, 2006, which claimspriority from U.S. Provisional Application 60/682,009, filed May 18,2005. The entire contents of each of the aforementioned applications areincorporated herein by reference.

BACKGROUND OF THE INVENTION

Granulocyte-macrophage colony stimulating factor, GM-CSF, was originallyidentified as a hemopoietic growth factor. It is produced by a number ofcell types including lymphocytes, monocytes, endothelial cells,fibroblasts and some malignant cells (Metcalf et al., 1986; Clark andKamen, 1987; Hart et al., 1988; Metcalf et al., 1986). In addition tohaving a function of growth stimulation and differentiation onhemopoietic precursor cells, GM-CSF also was discovered as having avariety of effects on cells of the immune system expressing the GM-CSFreceptor (for review see: Hamilton, 2002; de Groot et al., 1998). Themost important of these functions is the activation of monocytes,macrophages and granulocytes in several immune and inflammatoryprocesses (Gasson et al., 1990b; Gasson et al., 1990a; Hart et al.,1988; Rapoport et al., 1992).

Mature GM-CSF is a monomeric protein of 127 amino acids with twoglycosylation sites. The variable degree of glycosylation results in amolecular weight range between 14 kDa and 35 kDa. Non-glycosylated andglycosylated GM-CSF show similar activity in vitro (Cebon et al., 1990).The crystallographic analysis of GM-CSF revealed a barrel-shapedstructure composed of four short alpha helices (Diederichs et al.,1991). The overall folding is similar to other growth factors likegrowth hormone, interleukin-2 and interleukin-4.

GM-CSF exerts its biological activity by binding to its receptor(Kastelein and Shanafelt, 1993; Sisson and Dinarello, 1988). The mostimportant sites of GM-CSF receptor (GM-CSF-R) expression are on the cellsurface of myeloid cells and endothelial cells, whereas lymphocytes areGM-CSF-R negative. The native receptor is composed of at least twosubunits, alpha and beta. The alpha subunit imparts ligand specificityand binds GM-CSF with nanomolar affinity (Gearing et al., 1989; Gassonet al., 1986). The beta subunit is also part of the interleukin-3 andinterleukin-5 receptor complexes and, in association with the GM-CSFreceptor alpha subunit and GM-CSF, leads to the formation of a complexwith picomolar binding affinity (Hayashida et al., 1990). The bindingdomains on GM-CSF for the receptor have been mapped: GM-CSF interactswith the beta subunit of its receptor via a very restricted region inthe first alpha helix of GM-CSF (Shanafelt et al., 1991b, Shanafelt etal., 1991a, Lopez et al., 1991). Binding to the alpha subunit could bemapped to the third alpha helix, helix C, the initial residues of theloop joining helices C and D, and to the carboxyterminal tail of GM-CSF(Brown et al., 1994).

Formation of the GM-CSF trimeric receptor complex leads to theactivation of complex signaling cascades involving molecules of theJAK/STAT families, Shc, Ras, Raf, the MAP kinases,phosphatidylinositol-3-kinase and NFkB, finally leading to transcriptionof c-myc, c-fos and c-jun. Activation is mainly induced by the betasubunit of the receptor (Hayashida et al., 1990; Kitamura et al., 1991;Sato et al., 1993). The shared beta subunit is also responsible for theoverlapping functions exerted by IL-3, IL-5 and GM-CSF (for review see:de Groot et al., 1998).

Apart from its hemopoietic growth and differentiation stimulatingactivity, GM-CSF functions especially as a proinflammatory cytokine.Macrophages and monocytes as well as neutrophils and eosinophils becomeactivated by GM-CSF, resulting in the release of other cytokines andchemokines, matrix degrading proteases, increased HLA expression andincreased expression of cell adhesion molecules or receptors forCC-chemokines. The latter, in turn, leads to increased chemotaxis ofinflammatory cells into inflamed tissue (Chantry et al., 1990; Hamilton,2002; Sisson and Dinarello, 1988; Zhang et al., 1998; Hamilton et al.,1993; Lopez et al., 1986; Cheng et al., 2001; Gomez-Cambronero et al.,2003). Often, GM-CSF exerts its activity in synergy with otherinflammatory stimulating factors like other cytokines or LPS, e.g.neutrophils treated with GM-CSF in combination with e.g. LPS will showincreased oxidative burst (Kaufman et al., 1989; Rapoport et al., 1992).

GM-CSF as target for anti-inflammatory therapy:

Due to its diverse activating functions in the immune system, GM-CSF canbe considered as a target for anti-inflammatory therapy. Chronic andacute inflammatory diseases like rheumatoid arthritis (RA), multiplesclerosis (MS), Crohn's disease, psoriasis, asthma, atopic dermatitis orshock may well benefit from the blocking of GM-CSF activity andsubsequent reduction of harmful activities of GM-CSF responsive cells(Hamilton, 1993; Zhang et al., 1998; Hamilton, 2002).

Arthritis:

Several groups showed that GM-CSF, as well as its receptor, are presentin the synovial joint of arthritis patients (Alvaro-Gracia et al., 1991;Xu et al., 1989; Haworth et al., 1991). Additionally, GM-CSF was shownto cause flares of rheumatoid arthritis in patients treated with GM-CSFfor neutropenia in Felty's syndrome (Hazenberg et al., 1989) or afterchemotherapy (de Vries et al., 1991).

First hints on the usefulness of antibodies blocking GM-CSF for thetreatment of arthritis came from mouse in vivo studies (Campbell et al.,1997; Campbell et al., 1998; Cook et al., 2001). Specifically, Cook etal. showed that neutralizing antibodies to GM-CSF showed efficacy in acollagen-induced arthritis model. Blocking of GM-CSF led to a reductionof disease severity concerning inflammation, cartilage destruction andprogression of disease in initially affected limbs or progression toother limbs.

There are several effects of an anti-GM-CSF therapy from which thepatients with rheumatoid arthritis or with other inflammatory diseasescould benefit.

Blocking GM-CSF is expected to inhibit or reduce:

a) the activation and number of mature monocytes, macrophages, andneutrophils. Especially neutrophils and macrophages are abundant insynovial fluid and membrane. The macrophage number in the synovium hasbeen shown to correlate with the degree of erosion in RA joints(Mulherin et al., 1996; Burmester et al., 1997). Macrophages are thesource of a variety of other proinflammatory cytokines and matrixdegrading proteases. Production of H₂O₂ by neutrophils is part of thedestructive processes taking place in the arthritic joints (Babior,2000).

b) the differentiation of myeloid dendritic cells (DCs) and activationof synovial DCs (=synoviocytes). GM-CSF upregulates and maintains HLAclass II expression on DCs and RA synoviocytes (Alvaro-Gracia J M etal., 1991). DCs are instructed within the joint to acquire functionsassociated with the selective activation of inflammatory T-cells.Specific HLA-DR alleles have been linked to susceptibility to RA, andactivation of T-cells via antigen presentation of DC's may play acrucial role in this type of immune disease (Santiago-Schwarz et al.,2001).

Multiple Sclerosis:

In multiple sclerosis, elevated levels of GM-CSF correlate with theactive phase of MS (Carrieri et al., 1998; McQualter et al., 2001) andGM-CSF−/− mice fail to develop disease in the model system for MS,experimental encephalomyelitis, EAE (McQualter et al., 2001).

Asthma:

In asthma, increased amounts of GM-CSF in the lung have been reported(Broide and Firestein, 1991). At the same time eosinophils are elevated,on which GM-CSF in synergy with interleukin-5 acts in three ways: i) itstimulates the differentiation from progenitor cells into eosinophils,ii) it stimulates their functional activation, and iii) it prolongs thesurvival of eosinophils in the lung (Broide et al., 1992; Yamashita etal., 2002). Thus, reduction of the survival of eosinophils in asthmaticairways by blocking GM-CSF is likely to ameliorate disease. Theusefulness of anti-GM-CSF neutralizing antibodies was further shown in amodel for murine asthma where the administration of such antibodies ledto significant reduction of airway hyperresponsiveness and airwayinflammation (Yamashita et al., 2002).

In a different mouse model, LPS-dependent inflammation of the lung couldbe reduced by application of anti-GM-CSF antibody 22E9 in the mouse(Bozinovski et al., 2003).

Toxic Effects:

Mice homozygous for a disrupted granulocyte/macrophagecolony-stimulating factor (GM-CSF) gene develop normally and show nomajor perturbation of hematopoiesis up to 12 weeks of age. While mostGM-CSF-deficient mice are superficially healthy and fertile, all developa disorganized vascular extracellular matrix with disrupted and reducedcollagen bundles and abnormal lungs with impaired pulmonary surfactantclearance and reduced resistance to microbial pathogens in the lung.Features of the latter pathology resemble the human disorder pulmonaryalveolar proteinosis (PAP). GM-CSF does not seem to be essential for themaintenance of normal levels of the major types of mature hematopoieticcells and their precursors in blood, marrow, and spleen. However, theyimplicate GM-CSF as being essential for normal vascular development,pulmonary physiology, and for resistance to local infection (Stanley etal., 1994; Dranoff et al., 1994; Plenz et al., 2003; Shibata et al.,2001). Recently, a strong association of auto-antibodies to GM-CSF withPAP has additionally implicated GM-CSF signaling abnormalities in thepathogenesis of PAP in humans. Together, these observations demonstratethat GM-CSF has a critical role in regulation of surfactant homeostasisand alveolar macrophage innate immune functions in the lung (Bonfield etal., 2002; Trapnell and Whitsett, 2002; Uchida et al., 2004; Kitamura etal., 1999).

High titers of autoantibodies with blocking activity to GM-CSF have beendescribed in patients with myasthenia gravis. These patients did notshow any other autoimmune phenomena or hemopoietic deficiencies or“other obvious clinical correlates” (Meager et al., 1999).

The compound E21R, a modified form of GM-CSF that antagonizes thefunction of GM-CSF, had been evaluated in a phase I safety trial and wasfound to have a good safety profile in cancer patients (Olver et al.,2002).

Thus, apart from the lung function, which should be monitored closely,other side effects are not expected when applying an anti-GM-CSFtherapy.

So far, only antibodies derived from non-human species with GM-CSFneutralizing function have been generated. For example, EP 0499161 A1describes an antibody generated by immunization of mice witholigopeptides, the sequence of which is derived from a GM-CSF.Furthermore, the application discloses a method of alleviating in amammal in need thereof an undesirable effect of GM-CSF, which comprisesadministering to said mammal a GM-CSF-inhibiting amount of animmunoglobulin. However, that antibody is a murine antibody, renderingit unsuitable for human administration.

Additionally, WO 03/068920 discloses an inhibitory chimeric mouse/humanIgG1 antibody. Antibodies that contain non-human sequences are likely toelicit an immune response in the human patient and are not appropriatefor the therapeutic administration. For instance, in diseases wherelong-term treatment is required (e.g. chronic inflammatory diseases likerheumatoid arthritis, asthma and multiple sclerosis), continuedadministration of a non-human therapeutic agent increases the likelihoodof a severe inflammatory reaction and the production of human antibodiesthat may neutralize the therapeutic agent.

Correspondingly, in light of the great potential for anti-GM-CSFantibody therapy, there is a high need for human anti-GM-CSF antibodieswith high affinity that effectively block the GM-CSF/GM-CSF receptorinteraction. Additionally, it would be advantageous to have one or moreantibodies that can cross-react with GM-CSF of one or more non-humanspecies in order to test their efficacy in animal-based in vivo models.

The present invention satisfies these and other needs by providing fullyhighly efficacious anti-GM-CSF antibodies, which are described below.

SUMMARY OF THE INVENTION

It is an object of the invention to provide human and humanizedantibodies that can effectively block the GM-CSF/GM-CSF receptorinteraction.

It is another object of the invention to provide antibodies that aresafe for human administration.

It is also an object of the present invention to provide methods fortreating disease or and/or conditions associated with the presence ofGM-CSF by using one or more antibodies of the invention. These and otherobjects of the invention are more fully described herein.

In one aspect, the invention provides an antigen-binding region that isspecific for human GM-CSF, where the isolated human or humanizedantibody or functional fragment thereof is able (i) to block interactionof 0.5 μg/ml human GM-CSF with the alpha chain of human GM-CSF receptorexpressed on about 2×10⁵ CHO-K1 cells by at least 50% under thefollowing conditions: (a) the concentration of the human GM-CSF receptoralpha chain expressed on the CHO-K1 cells is similar to theconcentration of human GM-CSF receptor alpha chain expressed on about2×10⁵ CHO-GMRa#11 cells, and (b) the concentration of the isolated humanor humanized antibody or functional fragment thereof is about 5 μg/ml;and (ii) to neutralize 0.25 ng/ml human GM-CSF in a TF-1 proliferationassay with an at least five-fold lower IC₅₀ value than referenceantibody BVD2-21C11 and/or reference antibody MAB215. As used herein, a“TF-1 proliferation assay” is defined as the assay essentially asdescribed in Example 5B. The skilled worker can obtain CHO-K1 cellsexpressing human GM-CSF receptor alpha chain at a concentration similarto that which is expressed on about 2×10⁵ CHO-GMRa#11 cells by followingthe teachings provided herein.

The invention additionally provides an isolated human or humanizedantibody or functional antibody fragment that contains anantigen-binding region as disclosed herein. Such an antibody orfunctional fragment thereof may contain an antigen-binding region thatcontains an H-CDR3 region depicted in SEQ ID NO: 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 49, 50, 51 or 52; the antigen-binding region may furtherinclude an H-CDR2 region depicted in SEQ ID NO: 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 49, 50, 51 or 52; and the antigen-binding region alsomay contain an H-CDR1 region depicted in SEQ ID NO: 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 49, 50, 51 or 52. Such an antibody or functionalfragment thereof may contain an antigen-binding region that contains avariable heavy chain depicted in SEQ ID NO: 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 49, 50, 51 or 52. Such a GM-CSF-specific antibody of theinvention may contain an antigen-binding region that contains an L-CDR3region depicted in SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, 38, 39, 40,58, 59, 60 or 61; the antigen-binding region may further include anL-CDR2 region depicted in SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 58, 59, 60 or 61; and the antigen-binding region also may contain anL-CDR1 region depicted in SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 58, 59, 60 or 61. Such an antibody or functional fragment thereofmay contain an antigen-binding region that contains a variable lightchain depicted in SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 58,59, 60 or 61.

Peptide variants of the sequences disclosed herein are also embraced bythe present invention. Accordingly, the invention includes anti-GM-CSFantibodies having a heavy chain amino acid sequence with: at least 60percent sequence identity in the CDR regions with the CDR regionsdepicted in SEQ ID NO: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 49, 50,51 or 52; and or at least 80 percent sequence homology in the CDRregions with the CDR regions depicted in SEQ ID NO: 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 49, 50, 51 or 52. Further included are anti-GM-CSFantibodies having a light chain amino acid sequence with: at least 60percent sequence identity in the CDR regions with the CDR regionsdepicted in SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 58, 59,60 or 61; and or at least 80 percent sequence homology in the CDRregions with the CDR regions depicted in SEQ ID NO: 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 58, 59, 60 or 61.

An antibody of the invention may be an IgG (e.g., IgG₁), while anantibody fragment may be a Fab or scFv, for example. An inventiveantibody fragment, accordingly, may be, or may contain, anantigen-binding region that behaves in one or more ways as describedherein.

The invention also is related to isolated nucleic acid sequences, eachof which can encode an antigen-binding region of a human or humanizedantibody or a functional antibody fragment that is specific for GM-CSF.Such a nucleic acid sequence may encode a variable heavy chain of anisolated human or humanized antibody or functional fragment thereofcomprising SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 44, 45, 46, 47 or48, or a nucleic acid sequence that hybridizes under high stringencyconditions to the complementary strand of SEQ ID NO: 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 44, 45, 46, 47 or 48. The nucleic acid might encode avariable light chain of an isolated human or humanized antibody orfunctional fragment thereof comprising SEQ ID NO: 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 53, 54, 55, 56 or 57, or a nucleic acid sequencethat hybridizes under high stringency conditions to the complementarystrand of SEQ ID NO: 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 53, 54, 55,56 or 57.

The nucleic acid sequence might encode an antigen-binding region of ahuman or humanized antibody or a functional antibody fragment that isspecific for human GM-CSF, where the antibody or functional fragmentthereof is able (i) to block interaction of 0.5 μg/ml human GM-CSF withthe alpha chain of human GM-CSF receptor expressed on 2×10⁵ CHO-K1 cellsby at least 50% under the following conditions: (a) the concentration ofsaid human GM-CSF receptor alpha chain expressed on said CHO-K1 cells issimilar to the concentration of human GM-CSF receptor alpha chainexpressed on 2×10⁵ CHO-GMRa#11 cells and (b) the concentration of saidisolated human or humanized antibody or functional fragment thereof isabout 5 μg/ml, and (ii) to neutralize 0.25 ng/ml human GM-CSF in a TF-1proliferation assay with an at least five-fold lower IC₅₀ value than thereference antibody BVD2-21C11 and/or reference antibody MAB215.

Nucleic acids of the invention are suitable for recombinant production.Thus, the invention also relates to vectors and host cells containing anucleic acid sequence of the invention. Such host cells might bebacterial or eukaryotic cells.

Compositions of the invention may be used for therapeutic orprophylactic applications. The invention, therefore, includes apharmaceutical composition containing an inventive antibody (orfunctional antibody fragment) and a pharmaceutically acceptable carrieror excipient therefor. In a related aspect, the invention provides amethod for treating a disorder or condition associated with theundesired presence of GM-CSF or GM-CSF expressing cells. Such methodcontains the steps of administering to a subject in need thereof aneffective amount of the pharmaceutical composition that contains aninventive antibody as described or contemplated herein. Such a disorderor condition might be an inflammatory disease, such as rheumatoidarthritis, multiple sclerosis, Crohn's disease, psoriasis, asthma,atopic dermatitis and shock.

Human or humanized antibodies (and functional fragments thereof) of thepresent invention may be cross-reactive with rat and/or rhesus (macaca)GM-CSF, as determined by solution equilibrium titration (SET), and/orTF1 proliferation assay.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1a provides nucleic acid sequences of various novel antibodyvariable heavy chain regions.

FIG. 1b provides amino acid sequences of various novel antibody variableheavy chain regions. CDR regions HCDR1, HCDR2 and HCDR3 are designatedfrom N- to C-terminus in boldface.

FIG. 2a provides nucleic acid sequences of various novel antibodyvariable light chain regions.

FIG. 2b provides amino acid sequences of various novel antibody variablelight chain regions. CDR regions LCDR1, LCDR2 and LCDR3 are designatedfrom N- to C-terminus in boldface.

FIG. 3 provides amino acid sequences of variable heavy chain regions ofconsensus-based HuCAL® antibody master gene sequences. CDR regionsHCDR1, HCDR2 and HCDR3 are designated from N- to C-terminus in boldface.

FIG. 4 provides amino acid sequences of variable light chain regions ofconsensus-based HuCAL® antibody master gene sequences. CDR regionsLCDR1, LCDR2 and LCDR3 are designated from N- to C-terminus in boldface.

FIG. 5 provides an example of a DNA sequence of pMORPH®X9_MOR03929_FHexpression vector (SEQ ID NO: 43).

FIG. 6 provides expression level of GM-CSF receptor alpha, as determinedby FACS analysis using the GM-CSF receptor alpha specific antibodyMAB1006. CHO-GMRa#11 (solid line) is shown in comparison to CHO-K1(dotted line). The x-axis represents the relative fluorescence value(RFL), measured in FL2 channel; the y-axis represents cell count.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based on the discovery of novel antibodies thatare specific to or have a high affinity for GM-CSF and possess one ormore other novel properties. Preferably, an antibody of the inventioncan deliver a therapeutic benefit to a subject. The antibodies of theinvention, which may be human or humanized, can be used in manycontexts, which are more fully described herein.

A “human” antibody or functional human antibody fragment is herebydefined as one that is not chimeric (e.g., not “humanized”) and not from(either in whole or in part) a non-human species. A human antibody orfunctional antibody fragment can be derived from a human or can be asynthetic human antibody. A “synthetic human antibody” is defined hereinas an antibody having a sequence derived, in whole or in part, in silicofrom synthetic sequences that are based on the analysis of known humanantibody sequences. In silico design of a human antibody sequence orfragment thereof can be achieved, for example, by analyzing a databaseof human antibody or antibody fragment sequences and devising apolypeptide sequence utilizing the data obtained therefrom. Anotherexample of a human antibody or functional antibody fragment is one thatis encoded by a nucleic acid isolated from a library of antibodysequences of human origin (i.e., such library being based on antibodiestaken from a human natural source).

A “humanized antibody” or functional humanized antibody fragment isdefined herein as one that is (i) derived from a non-human source (e.g.,a transgenic mouse which bears a heterologous immune system), whichantibody is based on a human germline sequence; or (ii) chimeric,wherein the variable domain is derived from a non-human origin and theconstant domain is derived from a human origin or (iii) CDR-grafted,wherein the CDRs of the variable domain are from a non-human origin,while one or more frameworks of the variable domain are of human originand the constant domain (if any) is of human origin.

As used herein, an antibody “binds specifically to,” is “specificto/for” or “specifically recognizes” an antigen (here, GM-CSF) if suchantibody is able to discriminate between such antigen and one or morereference antigen(s), since binding specificity is not an absolute, buta relative property. In its most general form (and when no definedreference is mentioned), “specific binding” is referring to the abilityof the antibody to discriminate between the antigen of interest and anunrelated antigen, as determined, for example, in accordance with one ofthe following methods. Such methods comprise, but are not limited toWestern blots, ELISA-, RIA-, ECL-, IRMA-tests and peptide scans. Forexample, a standard ELISA assay can be carried out. The scoring may becarried out by standard color development (e.g. secondary antibody withhorseradish peroxide and tetramethyl benzidine with hydrogenperoxide).The reaction in certain wells is scored by the optical density, forexample, at 450 nm. Typical background (=negative reaction) may be 0.1OD; typical positive reaction may be 1 OD. This means the differencepositive/negative can be more than 10-fold. Typically, determination ofbinding specificity is performed by using not a single referenceantigen, but a set of about three to five unrelated antigens, such asmilk powder, BSA, transferrin or the like.

However, “specific binding” also may refer to the ability of an antibodyto discriminate between the target antigen and one or more closelyrelated antigen(s), which are used as reference points, e.g. betweenGM-CSF and IL3, IL5, IL-4, IL13 or M-CSF. Additionally, “specificbinding” may relate to the ability of an antibody to discriminatebetween different parts of its target antigen, e.g. different domains orregions of GM-CSF, or between one or more key amino acid residues orstretches of amino acid residues of GM-CSF.

Also, as used herein, an “immunoglobulin” (Ig) hereby is defined as aprotein belonging to the class IgG, IgM, IgE, IgA, or IgD (or anysubclass thereof), and includes all conventionally known antibodies andfunctional fragments thereof. A “functional fragment” of anantibody/immunoglobulin hereby is defined as a fragment of anantibody/immunoglobulin (e.g., a variable region of an IgG) that retainsthe antigen-binding region. An “antigen-binding region” of an antibodytypically is found in one or more hypervariable region(s) of anantibody, i.e., the CDR-1, -2, and/or -3 regions; however, the variable“framework” regions can also play an important role in antigen binding,such as by providing a scaffold for the CDRs. Preferably, the“antigen-binding region” comprises at least amino acid residues 4 to 103of the variable light (VL) chain and 5 to 109 of the variable heavy (VH)chain, more preferably amino acid residues 3 to 107 of VL and 4 to 111of VH, and particularly preferred are the complete VL and VH chains(amino acid positions 1 to 109 of VL and 1 to 113 of VH; numberingaccording to WO 97/08320). A preferred class of immunoglobulins for usein the present invention is IgG. “Functional fragments” of the inventioninclude the domain of a F(ab′)₂ fragment, a Fab fragment, scFv orconstructs comprising single immunoglobulin variable domains or singledomain antibody polypeptides, e.g. single heavy chain variable domainsor single light chain variable domains. The F(ab′)₂ or Fab may beengineered to minimize or completely remove the intermoleculardisulphide interactions that occur between the C_(H1) and C_(L)domains.

An antibody of the invention may be derived from a recombinant antibodylibrary that is based on amino acid sequences that have been designed insilico and encoded by nucleic acids that are synthetically created. Insilico design of an antibody sequence is achieved, for example, byanalyzing a database of human sequences and devising a polypeptidesequence utilizing the data obtained therefrom. Methods for designingand obtaining in silico-created sequences are described, for example, inKnappik et al., J. Mol. Biol. (2000) 296:57; Krebs et al., J. Immunol.Methods. (2001) 254:67; and U.S. Pat. No. 6,300,064 issued to Knappik etal., which hereby are incorporated by reference in their entirety.

Antibodies of the Invention

Throughout this document, reference is made to the followingrepresentative antibodies of the invention: “antibody nos.” or “MOR”03684, 04251, 03929, 04252, 04287, 04290, 04302, 04350, 04354, 04357,03682, 04283, 04297 and 04342. MOR03684 represents an antibody having avariable heavy region corresponding to SEQ ID NO: 1 (DNA)/SEQ ID NO: 11(protein) and a variable light region corresponding to SEQ ID NO: 21(DNA)/SEQ ID NO: 31 (protein). MOR04251 represents an antibody having avariable heavy region corresponding to SEQ ID NO: 2 (DNA)/SEQ ID NO: 12(protein) and a variable light region corresponding to SEQ ID NO: 22(DNA)/SEQ ID NO: 32 (protein). MOR03929 represents an antibody having avariable heavy region corresponding to SEQ ID NO: 3 (DNA)/SEQ ID NO: 13(protein) and a variable light region corresponding to SEQ ID NO: 23(DNA)/SEQ ID NO: 33 (protein). MOR04252 represents an antibody having avariable heavy region corresponding to SEQ ID NO: 4 (DNA)/SEQ ID NO: 14(protein) and a variable light region corresponding to SEQ ID NO: 24(DNA)/SEQ ID NO: 34 (protein). MOR04287 represents an antibody having avariable heavy region corresponding to SEQ ID NO: 5 (DNA)/SEQ ID NO: 15(protein) and a variable light region corresponding to SEQ ID NO: 25(DNA)/SEQ ID NO: 35 (protein). MOR04290 represents an antibody having avariable heavy region corresponding to SEQ ID NO: 6 (DNA)/SEQ ID NO: 16(protein) and a variable light region corresponding to SEQ ID NO: 26(DNA)/SEQ ID NO: 36 (protein). MOR04302 represents an antibody having avariable heavy region corresponding to SEQ ID NO: 7 (DNA)/SEQ ID NO: 17(protein) and a variable light region corresponding to SEQ ID NO: 27(DNA)/SEQ ID NO: 37 (protein). MOR04350 represents an antibody having avariable heavy region corresponding to SEQ ID NO: 8 (DNA)/SEQ ID NO: 18(protein) and a variable light region corresponding to SEQ ID NO: 28(DNA)/SEQ ID NO: 38 (protein). MOR04354 represents an antibody having avariable heavy region corresponding to SEQ ID NO: 9 (DNA)/SEQ ID NO: 19(protein) and a variable light region corresponding to SEQ ID NO: 29(DNA)/SEQ ID NO: 39 (protein). MOR04357 represents an antibody having avariable heavy region corresponding to SEQ ID NO: 10 or 48 (DNA)/SEQ IDNO: 20 (protein) and a variable light region corresponding to SEQ ID NO:30 or 57 (DNA)/SEQ ID NO: 40 (protein). MOR03682 represents an antibodyhaving a variable heavy region corresponding to SEQ ID NO: 44 (DNA)/SEQID NO: 49 (protein) and a variable light region corresponding to SEQ IDNO: 53 (DNA)/SEQ ID NO: 58 (protein). MOR04283 represents an antibodyhaving a variable heavy region corresponding to SEQ ID NO: 45 (DNA)/SEQID NO: 50 (protein) and a variable light region corresponding to SEQ IDNO: 54 (DNA)/SEQ ID NO: 59 (protein). MOR04297 represents an antibodyhaving a variable heavy region corresponding to SEQ ID NO: 46 (DNA)/SEQID NO: 51 (protein) and a variable light region corresponding to SEQ IDNO: 55 (DNA)/SEQ ID NO: 60 (protein). MOR04342 represents an antibodyhaving a variable heavy region corresponding to SEQ ID NO: 47 (DNA)/SEQID NO: 52 (protein) and a variable light region corresponding to SEQ IDNO: 56 (DNA)/SEQ ID NO: 61 (protein).

In one aspect, the invention provides antibodies having anantigen-binding region that can bind specifically to or has a highaffinity for GM-CSF. An antibody is said to have a “high affinity” foran antigen if the affinity measurement is at least 100 nM (monovalentaffinity of Fab fragment). An inventive antibody or antigen-bindingregion preferably can bind to GM-CSF with an affinity of about less than100 nM, more preferably less than about 60 nM, and still more preferablyless than about 30 nM. Further preferred are antibodies that bind toGM-CSF with an affinity of less than about 10 nM, and more preferablyless than about 3 nM. For instance, the affinity of an antibody of theinvention against GM-CSF may be about 10.0 nM or 1 pM (monovalentaffinity of Fab fragment).

Table 1 provides a summary of affinities of representative antibodies ofthe invention, as determined by surface plasmon resonance (Biacore) andSolution Equilibrium Titration (SET) analysis:

TABLE 1 Antibody Affinities Biacore SET MOR0 K_(D) (pM) K_(D) (pM) 36846420 16000 4251 70 7.4 3929 4260 2000 4302 174 63.5 4287 nd 17.9 4252 556 4290 122 11.1 4350 19 1.1 4354 21 2.8 4357 7 0.4 3682 nd 11406 4283 nd113 4297 nd 49.2 4342 nd 4.9 “nd”: not determined

With reference to Table 1, the affinity of MOR03684, 04251, 03929,04252, 04357, 04290, 04302, 04350 and 04354 was measured by surfaceplasmon resonance (Biacore) on immobilized recombinant GM-CSF. The Fabformat of MOR03684, 04251, 03929, 04252, 04357, 04290, 04302, 04350 and04354 exhibit a monovalent affinity range between about 6420 and 7 pM.

The Fab format was also used for the determination of the affinities bysolution equilibrium titration (SET). The right column of Table 1denotes the binding strength of between about 16000 and 0.4 pM of theMORs in this method.

An antibody of the invention preferably is species cross-reactive withhumans and at least one other species, which may be a rodent species ora non-human primate. The non-human primate can be rhesus. The rodentspecies can be rat. An antibody that is cross reactive with at least onerodent species, for example, can provide greater flexibility andbenefits over known anti-GM-CSF antibodies, for purposes of conductingin vivo studies in multiple species with the same antibody.

Preferably, an antibody of the invention not only is able to bind toGM-CSF, but also is able to block the interaction of human GM-CSF withthe alpha chain of human GM-CSF receptor expressed on CHO-K1 cells by atleast 25%, preferably by at least 50%, more preferably by at least 60%,more preferably by at least 70%, preferably by at least 85% and mostpreferably by at least 100%. In a preferred embodiment, an antibody ofthe invention is able to block interaction of 0.5 μg/ml human GM-CSFwith the alpha chain of human GM-CSF receptor expressed on about 2×10⁵CHO-K1 cells by at least 50% under the following conditions: theconcentration of the human GM-CSF receptor alpha chain expressed on theCHO-K1 cells is similar to the concentration of human GM-CSF receptoralpha chain expressed on about 2×10⁵ CHO-GMRa#11 cells, and theconcentration of the inventive antibody is about 5 μg/ml.

In this regard, the skilled worker can obtain CHO-K1 cells expressinghuman GM-CSF receptor alpha at a concentration similar to that which isexpressed on about 2×10⁵ CHO-GMRa#11 cells by, e.g., by transfecting apopulation of CHO-K1 cells with a suitable expression vector encodingGM-CSF receptor alpha to generate different stable cell lines expressingdefined levels GM-CSF receptor alpha; then, the stable cell lines areanalyzed in FACS analysis to determine GM-CSF receptor alpha expressionlevels according to the protocol essentially as described in Example 3C;a cell line that expresses human GM-CSF receptor alpha at aconcentration similar to that which is expressed on about 2×10⁵CHO-GMRa#11 cells is identified by comparing the median fluorescencevalue (MFL) of such transfected cells to the MFL value set forth inExample 3C. As used herein, a cell line is defined as expressing GM-CSFreceptor alpha at a concentration “similar” to that which is expressedon about 2×10⁵ CHO-GMRa#11 cells” if the MFL value of the transfectedcell line does not deviate by more than a two-fold factor from the MFLvalue for the CHO-GMRa#11 cell as set forth in Example 3C.

Furthermore, an antibody of the invention is able to neutralize humanGM-CSF in a TF-1 proliferation assay with a lower IC₅₀ value than thereference antibody BVD2-21C11 and/or MAB215, preferably an at leastfive-fold lower IC₅₀ value, more preferably with an at least 10-foldlower IC₅₀ value than the reference antibody BVD2-21C11 and/or MAB215,more preferably with an at least 15-fold lower IC₅₀ value than thereference antibody BVD2-21C11 and/or MAB215, more preferably with an atleast 20-fold lower IC₅₀ value than the reference antibody BVD2-21C11and/or MAB215, more preferably with an at least 30-fold lower IC₅₀ valuethan the reference antibody BVD2-21C11 and/or MAB215, more preferablywith an at least 50-fold lower IC₅₀ value than the reference antibodyBVD2-21C11 and/or MAB215, more preferably with an at least 100-foldlower IC₅₀ value than the reference antibody BVD2-21C11 and/or MAB215and most preferably with an at least 120-fold lower IC₅₀ value than thereference antibody BVD2-21C11 and/or MAB215.

Peptide Variants

Antibodies of the invention are not limited to the specific peptidesequences provided herein. Rather, the invention also embodies variantsof these polypeptides. With reference to the instant disclosure andconventionally available technologies and references, the skilled workerwill be able to prepare, test and utilize functional variants of theantibodies disclosed herein, while appreciating that variants having theability to block the interaction of GM-CSF to the alpha chain of theGM-CSF receptor fall within the scope of the present invention. As usedin this context, “ability to block the interaction of GM-CSF to thealpha chain of the GM-CSF receptor” means a functional characteristicascribed to an anti-GM-CSF antibody of the invention.

A variant can include, for example, an antibody that has at least onealtered complementarity determining region (CDR) (hyper-variable) and/orframework (FR) (variable) domain/position, vis-à-vis a peptide sequencedisclosed herein. To better illustrate this concept, a brief descriptionof antibody structure follows.

An antibody is composed of two peptide chains, each containing one(light chain) or three (heavy chain) constant domains and a variableregion (VL, VH), the latter of which is in each case made up of four FRregions and three interspaced CDRs. The antigen-binding site is formedby one or more CDRs, yet the FR regions provide the structural frameworkfor the CDRs and can also play an important role in antigen binding. Byaltering one or more amino acid residues in a CDR or FR region, theskilled worker routinely can generate mutated or diversified antibodysequences, which can be screened against the antigen, for new orimproved properties, for example.

Tables 2a (VH) and 2b (VL) delineate the CDR and FR regions for certainantibodies of the invention and compare amino acids at a given positionto each other and to corresponding consensus or “master gene” sequences(as described in U.S. Pat. No. 6,300,064):

The original HuCAL® master genes have been constructed with theirauthentic N-termini, e.g. VL lambda 3 contains the amino acids “SY” atposition 1 and 2; and VH3 contains the amino acid “E” at position 1.During construction of the HuCAL® Fab libraries, including the HuCALGOLD® library, the first two amino acids have been changed to “DI” inthe VL lambda 3 chain; and the first amino acid has been changed to “Q”in the VH3 chain.

The skilled worker can use the data in Tables 2a and 2b to designpeptide variants that are within the scope of the present invention. Itis preferred that variants are constructed by changing amino acidswithin one or more CDR regions; a variant might also have one or morealtered framework regions. With reference to a comparison of the novelantibodies to each other, candidate residues that can be changed includee.g. residues 27 or 51 of the variable light and e.g. residues 32 or 56of the variable heavy chains of MOR04251, since these are positions ofvariance vis-á-vis each other. Alterations also may be made in theframework regions. For example, a peptide FR domain might be alteredwhere there is a deviation in a residue compared to a germline sequence.

With reference to a comparison of the novel antibodies to thecorresponding consensus or “master gene” sequence, candidate residuesthat can be changed include e.g. residues 27, 50 or 90 of the variablelight chain of MOR04251 compared to VLλ3 and e.g. residues 33, 52 or 96of the variable heavy chain of MOR04251 compared to VH3. Alternatively,the skilled worker could make the same analysis by comparing the aminoacid sequences disclosed herein to known sequences of the same class ofsuch antibodies, using, for example, the procedure described by Knappiket al. (2000), and U.S. Pat. No. 6,300,064 issued to Knappik et al.Furthermore, variants may be obtained by using one MOR as starting pointfor optimization by diversifying one or more amino acid residues in theMOR, preferably amino acid residues in one or more CDRs, and byscreening the resulting collection of antibody variants for variantswith improved properties. Particularly preferred is diversification ofone or more amino acid residues in CDR-3 of VL, CDR-3 of VH, CDR-1 of VLand/or CDR-2 of VH. Diversification can be done by synthesizing acollection of DNA molecules using trinucleotide mutagenesis (TRIM)technology (Vimekäs et al., 1994).

Conservative Amino Acid Variants

Polypeptide variants may be made that conserve the overall molecularstructure of an antibody peptide sequence described herein. Given theproperties of the individual amino acids, some rational substitutionswill be recognized by the skilled worker. Amino acid substitutions,i.e., “conservative substitutions,” may be made, for instance, on thebasis of similarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.

For example, (a) nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; (b) polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; (c) positivelycharged (basic) amino acids include arginine, lysine, and histidine; and(d) negatively charged (acidic) amino acids include aspartic acid andglutamic acid. Substitutions typically may be made within groups(a)-(d). In addition, glycine and proline may be substituted for oneanother based on their ability to disrupt α-helices. Similarly, certainamino acids, such as alanine, cysteine, leucine, methionine, glutamicacid, glutamine, histidine and lysine are more commonly found inα-helices, while valine, isoleucine, phenylalanine, tyrosine, tryptophanand threonine are more commonly found in β-pleated sheets. Glycine,serine, aspartic acid, asparagine, and proline are commonly found inturns. Some preferred substitutions may be made among the followinggroups: (i) S and T; (ii) P and G; and (iii) A, V, L and I. Given theknown genetic code, and recombinant and synthetic DNA techniques, theskilled scientist readily can construct DNAs encoding the conservativeamino acid variants.

As used herein, “sequence identity” between two polypeptide sequences,indicates the percentage of amino acids that are identical between thesequences. “Sequence homology”, indicates the percentage of amino acidsthat either are identical or that represent conservative amino acidsubstitutions. Preferred polypeptide sequences of the invention have asequence identity in the CDR regions of at least 60%, more preferably,at least 70% or 80%, still more preferably at least 90% and mostpreferably at least 95%. Preferred antibodies also have a sequencehomology in the CDR regions of at least 80%, more preferably 90% andmost preferably 95%.

DNA Molecules of the Invention

The present invention also relates to the DNA molecules that encode anantibody of the invention. These sequences include, but are not limitedto, those DNA molecules set forth in FIGS. 1a and 2 a.

DNA molecules of the invention are not limited to the sequencesdisclosed herein, but also include variants thereof. DNA variants withinthe invention may be described by reference to their physical propertiesin hybridization. The skilled worker will recognize that DNA can be usedto identify its complement and, since DNA is double stranded, itsequivalent or homolog, using nucleic acid hybridization techniques. Italso will be recognized that hybridization can occur with less than 100%complementarity. However, given appropriate choice of conditions,hybridization techniques can be used to differentiate among DNAsequences based on their structural relatedness to a particular probe.For guidance regarding such conditions see, Sambrook et al., 1989(Sambrook, J., Fritsch, E. F. and Maniatis, T. (1989) Molecular Cloning:A laboratory manual, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, USA) and Ausubel et al., 1995 (Ausubel, F. M., Brent, R.,Kingston, R. E., Moore, D. D., Sedman, J. G., Smith, J. A., & Struhl, K.eds. (1995). Current Protocols in Molecular Biology. New York: JohnWiley and Sons).

Structural similarity between two polynucleotide sequences can beexpressed as a function of “stringency” of the conditions under whichthe two sequences will hybridize with one another. As used herein, theterm “stringency” refers to the extent that the conditions disfavorhybridization. Stringent conditions strongly disfavor hybridization, andonly the most structurally related molecules will hybridize to oneanother under such conditions. Conversely, non-stringent conditionsfavor hybridization of molecules displaying a lesser degree ofstructural relatedness. Hybridization stringency, therefore, directlycorrelates with the structural relationships of two nucleic acidsequences. The following relationships are useful in correlatinghybridization and relatedness (where T_(m) is the melting temperature ofa nucleic acid duplex):

-   -   a. T_(m)=69.3+0.41(G+C) %    -   b. The T_(m) of a duplex DNA decreases by 1° C. with every        increase of 1% in the number of mismatched base pairs.    -   C. (T_(m))_(μ2)−(T_(m))_(μ1)=18.5 log₁₀μ2/μ1        -   where μ1 and μ2 are the ionic strengths of two solutions.

Hybridization stringency is a function of many factors, includingoverall DNA concentration, ionic strength, temperature, probe size andthe presence of agents which disrupt hydrogen bonding. Factors promotinghybridization include high DNA concentrations, high ionic strengths, lowtemperatures, longer probe size and the absence of agents that disrupthydrogen bonding. Hybridization typically is performed in two phases:the “binding” phase and the “washing” phase.

First, in the binding phase, the probe is bound to the target underconditions favoring hybridization. Stringency is usually controlled atthis stage by altering the temperature. For high stringency, thetemperature is usually between 65° C. and 70° C., unless short (<20 nt)oligonucleotide probes are used. A representative hybridization solutioncomprises 6×SSC, 0.5% SDS, 5×Denhardt's solution and 100 μg ofnonspecific carrier DNA. See Ausubel et al., section 2.9, supplement 27(1994). Of course, many different, yet functionally equivalent, bufferconditions are known. Where the degree of relatedness is lower, a lowertemperature may be chosen. Low stringency binding temperatures arebetween about 25° C. and 40° C. Medium stringency is between at leastabout 40° C. to less than about 65° C. High stringency is at least about65° C.

Second, the excess probe is removed by washing. It is at this phase thatmore stringent conditions usually are applied. Hence, it is this“washing” stage that is most important in determining relatedness viahybridization. Washing solutions typically contain lower saltconcentrations. One exemplary medium stringency solution contains 2×SSCand 0.1% SDS. A high stringency wash solution contains the equivalent(in ionic strength) of less than about 0.2×SSC, with a preferredstringent solution containing about 0.1×SSC. The temperatures associatedwith various stringencies are the same as discussed above for “binding.”The washing solution also typically is replaced a number of times duringwashing. For example, typical high stringency washing conditionscomprise washing twice for 30 minutes at 55° C. and three times for 15minutes at 60° C.

Accordingly, the present invention includes nucleic acid molecules thathybridize to the molecules of set forth in FIGS. 1a and 2a under highstringency binding and washing conditions, where such nucleic moleculesencode an antibody or functional fragment thereof having properties asdescribed herein. Preferred molecules (from an mRNA perspective) arethose that have at least 75% or 80% (preferably at least 85%, morepreferably at least 90% and most preferably at least 95%) homology orsequence identity with one of the DNA molecules described herein.

Functionally Equivalent Variants

It is recognized that variants of DNA molecules provided herein can beconstructed in several different ways. For example, they may beconstructed as completely synthetic DNAs. Methods of efficientlysynthesizing oligonucleotides in the range of 20 to about 150nucleotides are widely available. See Ausubel et al., section 2.11,Supplement 21 (1993). Overlapping oligonucleotides may be synthesizedand assembled in a fashion first reported by Khorana et al., J. Mol.Biol. 72:209-217 (1971); see also Ausubel et al., supra, Section 8.2.Synthetic DNAs preferably are designed with convenient restriction sitesengineered at the 5′ and 3′ ends of the gene to facilitate cloning intoan appropriate vector.

As indicated, a method of generating variants is to start with one ofthe DNAs disclosed herein and then to conduct site-directed mutagenesis.See Ausubel et al., supra, chapter 8, Supplement 37 (1997). In a typicalmethod, a target DNA is cloned into a single-stranded DNA bacteriophagevehicle. Single-stranded DNA is isolated and hybridized with anoligonucleotide containing the desired nucleotide alteration(s). Thecomplementary strand is synthesized and the double stranded phage isintroduced into a host. Some of the resulting progeny will contain thedesired mutant, which can be confirmed using DNA sequencing. Inaddition, various methods are available that increase the probabilitythat the progeny phage will be the desired mutant. These methods arewell known to those in the field and kits are commercially available forgenerating such mutants.

Recombinant DNA Constructs and Expression

The present invention further provides recombinant DNA constructscomprising one or more of the nucleotide sequences of the presentinvention. The recombinant constructs of the present invention are usedin connection with a vector, such as a plasmid, phagemid, phage or viralvector, into which a DNA molecule encoding an antibody of the inventionis inserted.

The encoded gene may be produced by techniques described in Sambrook etal., 1989, and Ausubel et al., 1989. Alternatively, the DNA sequencesmay be chemically synthesized using, for example, synthesizers. See, forexample, the techniques described in OLIGONUCLEOTIDE SYNTHESIS (1984,Gait, ed., IRL Press, Oxford), which is incorporated by reference hereinin its entirety. Recombinant constructs of the invention are comprisedwith expression vectors that are capable of expressing the RNA and/orprotein products of the encoded DNA(s). The vector may further compriseregulatory sequences, including a promoter operably linked to the openreading frame (ORF). The vector may further comprise a selectable markersequence. Specific initiation and bacterial secretory signals also maybe required for efficient translation of inserted target gene codingsequences.

The present invention further provides host cells containing at leastone of the DNAs of the present invention. The host cell can be virtuallyany cell for which expression vectors are available. It may be, forexample, a higher eukaryotic host cell, such as a mammalian cell, alower eukaryotic host cell, such as a yeast cell or a prokaryotic cell,such as a bacterial cell. Introduction of the recombinant construct intothe host cell can be effected by calcium phosphate transfection, DEAE,dextran mediated transfection, electroporation or phage infection.

Bacterial Expression

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and, if desirable, to provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus.

Bacterial vectors may be, for example, bacteriophage-, plasmid- orphagemid-based. These vectors can contain a selectable marker andbacterial origin of replication derived from commercially availableplasmids typically containing elements of the well known cloning vectorpBR322 (ATCC 37017). Following transformation of a suitable host strainand growth of the host strain to an appropriate cell density, theselected promoter is de-repressed/induced by appropriate means (e.g.,temperature shift or chemical induction) and cells are cultured for anadditional period. Cells are typically harvested by centrifugation,disrupted by physical or chemical means, and the resulting crude extractretained for further purification.

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the proteinbeing expressed. For example, when a large quantity of such a protein isto be produced, for the generation of antibodies or to screen peptidelibraries, for example, vectors which direct the expression of highlevels of fusion protein products that are readily purified may bedesirable.

Therapeutic Methods

Therapeutic methods involve administering to a subject in need oftreatment a therapeutically effective amount of an antibody contemplatedby the invention. A “therapeutically effective” amount hereby is definedas the amount of an antibody that is of sufficient quantity toeffectively block the interaction between GM-CSF and its receptor in atreated area of a subject—either as a single dose or according to amultiple dose regimen, alone or in combination with other agents, whichleads to the alleviation of an adverse condition, yet which amount istoxicologically tolerable. The subject may be a human or non-humananimal (e.g., rat or rhesus).

An antibody of the invention might be co-administered with knownmedicaments, and in some instances the antibody might itself bemodified. For example, an antibody could be conjugated to an immunotoxinor radioisotope to potentially further increase efficacy.

The inventive antibodies can be used as a therapeutic or a diagnostictool in a variety of situations where GM-CSF is undesirably expressed orfound. Disorders and conditions particularly suitable for treatment withan antibody of the inventions are inflammatory diseases such asrheumatoid arthritis (RA), multiple sclerosis, Crohn's disease,psoriasis, asthma, atopic dermatitis or shock.

To treat any of the foregoing disorders, pharmaceutical compositions foruse in accordance with the present invention may be formulated in aconventional manner using one or more physiologically acceptablecarriers or excipients. An antibody of the invention can be administeredby any suitable means, which can vary, depending on the type of disorderbeing treated. Possible administration routes include parenteral (e.g.,intramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous), intrapulmonary and intranasal, and, if desired for localimmunosuppressive treatment, intralesional administration. In addition,an antibody of the invention might be administered by pulse infusion,with, e.g., declining doses of the antibody. Preferably, the dosing isgiven by injections, most preferably intravenous or subcutaneousinjections, depending in part on whether the administration is brief orchronic. The amount to be administered will depend on a variety offactors such as the clinical symptoms, weight of the individual, whetherother drugs are administered. The skilled artisan will recognize thatthe route of administration will vary depending on the disorder orcondition to be treated.

Determining a therapeutically effective amount of the novel polypeptide,according to this invention, largely will depend on particular patientcharacteristics, route of administration, and the nature of the disorderbeing treated. General guidance can be found, for example, in thepublications of the International Conference on Harmonisation and inREMINGTON'S PHARMACEUTICAL SCIENCES, chapters 27 and 28, pp. 484-528(18th ed., Alfonso R. Gennaro, Ed., Easton, Pa.: Mack Pub. Co., 1990).More specifically, determining a therapeutically effective amount willdepend on such factors as toxicity and efficacy of the medicament.Toxicity may be determined using methods well known in the art and foundin the foregoing references. Efficacy may be determined utilizing thesame guidance in conjunction with the methods described below in theExamples.

Diagnostic Methods

GM-CSF is expressed by various cell types including lymphocytes,monocytes, endothelial cells, fibroblasts and some malignant cells;thus, an anti-GM-CSF antibody of the invention may be employed in orderto image or visualize a site of possible accumulation of GM-CSF indifferent tissues in a patient. In this regard, an antibody can bedetectably labeled, through the use of radioisotopes, affinity labels(such as biotin, avidin, etc.) fluorescent labels, paramagnetic atoms,etc. Procedures for accomplishing such labeling are well known to theart. Clinical application of antibodies in diagnostic imaging arereviewed by Grossman, H. B., Urol. Clin. North Amer. 13:465-474 (1986)),Unger, E. C. et al., Invest. Radiol. 20:693-700 (1985)), and Khaw, B. A.et al., Science 209:295-297 (1980)).

The detection of foci of such detectably labeled antibodies might beindicative of a site of inflammation, for example. In one embodiment,this examination is done by removing samples of tissue or blood andincubating such samples in the presence of the detectably labeledantibodies. In a preferred embodiment, this technique is done in anon-invasive manner through the use of magnetic imaging, fluorography,etc. Such a diagnostic test may be employed in monitoring the success oftreatment of diseases, where presence or absence of GM-CSF is a relevantindicator. The invention also contemplates the use of an anti-GM-CSFantibody, as described herein for diagnostics in an ex vivo setting.

Therapeutic and Diagnostic Compositions

The antibodies of the present invention can be formulated according toknown methods to prepare pharmaceutically useful compositions, whereinan antibody of the invention (including any functional fragment thereof)is combined in a mixture with a pharmaceutically acceptable carriervehicle. Suitable vehicles and their formulation are described, forexample, in REMINGTON'S PHARMACEUTICAL SCIENCES (18th ed., Alfonso R.Gennaro, Ed., Easton, Pa.: Mack Pub. Co., 1990). In order to form apharmaceutically acceptable composition suitable for effectiveadministration, such compositions will contain an effective amount ofone or more of the antibodies of the present invention, together with asuitable amount of carrier vehicle.

Preparations may be suitably formulated to give controlled-release ofthe active compound. Controlled-release preparations may be achievedthrough the use of polymers to complex or absorb anti-GM-CSF antibody.The controlled delivery may be exercised by selecting appropriatemacromolecules (for example polyesters, polyamino acids, polyvinyl,pyrrolidone, ethylenevinyl-acetate, methylcellulose,carboxymethylcellulose, or protamine, sulfate) and the concentration ofmacromolecules as well as the methods of incorporation in order tocontrol release. Another possible method to control the duration ofaction by controlled release preparations is to incorporate anti-GM-CSFantibody into particles of a polymeric material such as polyesters,polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetatecopolymers. Alternatively, instead of incorporating these agents intopolymeric particles, it is possible to entrap these materials inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatine-microcapsules and poly(methylmethacylate) microcapsules,respectively, or in colloidal drug delivery systems, for example,liposomes, albumin microspheres, microemulsions, nanoparticles, andnanocapsules or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences (1980).

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampules, orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compositions may, if desired, be presented in a pack or dispenserdevice, which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

The invention further is understood by reference to the followingworking examples, which are intended to illustrate and, hence, not limitthe invention.

Examples Example 1: Generation of Human GM-CSF Specific Antibodies fromthe HuCAL GOLD® Library

A. Phagemid rescue, phage amplification and purification HuCAL GOLD®library was amplified in 2×YT medium containing 34 μg/ml chloramphenicoland 1% glucose (2×YT-CG). After helper phage infection (VCSM13) at anOD₆₀₀ of 0.5 (30 min at 37° C. without shaking; 30 min at 37° C. shakingat 250 rpm), cells were spun down (4120 g; 5 min; 4° C.), resuspended in2×YT/34 μg/ml chloramphenicol/50 μg/ml kanamycin/0.25 mM IPTG and grownovernight at 22° C. Phages were PEG-precipitated from the supernatant,resuspended in PBS/20% glycerol and stored at −80° C. Phageamplification between two panning rounds was conducted as follows:mid-log phase E. coli TG1 cells were infected with eluted phages andplated onto LB-agar supplemented with 1% of glucose and 34 μg/ml ofchloramphenicol. After overnight incubation at 30° C., colonies werescraped off and used to inoculate 2×YT-CG until an OD_(600nm) of 0.5 wasreached and helper phage added as described above.

B. Pannings with HuCAL GOLD®

For the selection of antibodies recognizing human GM-CSF several panningstrategies were applied. In summary, HuCAL GOLD® antibody-phages weredivided into three pools comprising different VH master genes. Thesepools were individually subjected to either a) a solid phase panning onbiotinylated human GM-CSF protein (custom made by R&D Systems,Minneapolis, Minn.) directly coated on neutravidin coated 96 well plates(Pierce, Rockford, Ill.) as solid support for three rounds or b) asolution panning on biotinylated human GM-CSF protein captured ontostreptavidin coated Dynabeads (Dynal, Oslo, Norway) for three rounds.

In detail, for panning on immobilized biotinylated GM-CSF, wells of theneutravidin plate were washed three times with 300 μl PBS. The antigenwas diluted to a concentration of 3 μg/ml (200 nM) in PBS and 0.1 ml wascoated per well for 2 h at room temperature. After two washing stepswith 300 μl PBS the wells were incubated with blocking buffer containing2× Chemiblocker (Chemicon, Temecula, Calif.) diluted 1:1 in PBS.

Prior to the selections, 100 μl of HuCAL GOLD® phages were pre-adsorbedin 100 μl blocking buffer containing 0.4 μl 25% Tween20 for 0.5 h at RT.Blocked phages were transferred in 100 μl aliquots to wells of aneutravidin plate for 0.5 h at RT. This step was repeated twice forpre-absorption.

After washing (2×300 μl PBS) of the coated and blocked neutravidinmicrotiter plate, 0.1 ml of the pre-adsorbed phages were added to thecoated wells and incubated for 1.5 h at RT shaking gently. Thisincubation was followed by 10 wash cycles with PBS/0.05% Tween20 at RT.

Bound phages were eluted by adding 120 μl of 20 mM DTT in 10 mM Tris pH8.0 per well for 10 min at RT. The eluate was removed and added to 14 mlE. coli TG1 grown to an OD_(600nm) of 0.6-0.8. Wells were additionallywashed with 200 μl PBS and this solution was also added to the TG1cells. Phage infection of E. coli was allowed for 45 min at 37° C.without shaking. Additionally, 200 μl of TG1 cells grown to anOD_(600nm) of 0.6-0.8 were added to the selection wells for 45 minutesat 37° C. without shaking. These TG-1 cells were added to the 14 mlculture already containing the phages from the first elution step. Aftercentrifugation for 10 min at 5000 rpm, the bacterial pellets were eachresuspended in 500 μl 2×YT medium, plated on 2×YT-CG agar plates andincubated overnight at 30° C. Colonies were then scraped from the platesand phages were rescued and amplified as described above.

The second and third rounds of selection were performed in an identicalway to the first round of selection with the only difference that thewashing conditions after binding of phage were more stringent.Additionally, in the third round of selection, phages were submitted toan additional preadsorption step on streptavidin-coated beads (DynabeadsM-280; Dynal). Eppendorf tubes were blocked with Chemiblocker solutionby incubation for 30 min at RT. Of each phage pool 0.3 ml were mixed 1:1with 2×Chemiblocker solution containing 0.05% Tween20 and incubated for1 h at RT in the blocked Eppendorf tubes on a rotator. Blocked phageswere then transferred to newly blocked Eppendorf tubes and 50 μl ofDynabeads M-280 were added for another 30 min for preadsorption. Beadswere removed using a magnetic device (Dynal MPC-E). Aliquots of 150 μlof phages were then transferred to neutravidin plates for furtherpreadsorption as in round 1 and 2 (see above).

For the solution panning using biotinylated GM-CSF coupled to Dynabeadsthe following protocol was applied: 1.5 ml Eppendorf tubes were blockedwith 1.5 ml 2×Chemiblocker diluted 1:1 with PBS over night at 4° C. 200μl streptavidin coated magnetic beads (Dynabeads M-280; Dynal) werewashed 1× with 200 μl PBS and resuspended in 200 μl 1×Chemiblocker(diluted in 1×PBS). Blocking of beads was performed in preblocked tubesover night at 4° C. Phages diluted in 500 μl PBS for each panningcondition were mixed with 500 μl 2×Chemiblocker/0.1% Tween 1 h at RT(rotator). Preadsorption of phages was performed twice: 50 μl of blockedStreptavidin magnetic beads were added to the blocked phages andincubated for 30 min at RT on a rotator. After separation of beads via amagnetic device (Dyna) MPC-E) the phage supernatant (˜1 ml) wastransferred to a new blocked tube and preadsorption was repeated on 50μl blocked beads for 30 min. Then, 200 nM biotinylated hGM-CSF was addedto blocked phages in a new blocked 1.5 ml tube and incubated for 1 h atRT on a rotator. 100 μl of blocked streptavidin magnetic beads wereadded to each panning phage pool and incubated 10 min at RT on arotator. Phage bound to biotinylated GM-CSF and therefore immobilized tothe magnetic beads were collected with a magnetic particle separator(Dyna) MPC-E). Beads were then washed 7× in PBS/0.05% Tween using arotator, followed by washing another three times with PBS. Elution ofphage from the Dynabeads was performed adding 300 μl 20 mM DTT in 10 mMTris/HCl pH8 to each tube for 10 min. Dynabeads were removed by themagnetic particle separator and the supernatant was added to 14 ml of aE. coli TG-1 culture grown to OD_(600nm) of 0.6-0.8. Beads were thenwashed once with 200 μl PBS and PBS containing additional removed phagewas added to the 14 ml E. coli TG-1 culture.

After centrifugation for 10 min at 5000 rpm, the bacterial pellets wereeach resuspended in 500 μl 2×YT medium, plated on 2×YT-CG agar platesand incubated overnight at 30° C. Colonies were then scraped from theplates and phages were rescued and amplified as described above.

The second and third rounds of the solution panning on biotinylatedGM-CSF was performed according to the protocol of the first round exceptfor increasing the stringency of the washing procedure.

C. Subcloning of Selected Fab Fragments and Expression of Soluble FabFragments

The Fab encoding inserts of the selected HuCAL GOLD® phagemids weresubcloned into the expression vector pMORPH®X9_Fab_FH (FIG. 5) tofacilitate rapid expression of soluble Fab. The DNA of the selectedclones was digested with XbaI and EcoRI, thereby cutting out the Fabencoding insert (ompA-VLCL and phoA-Fd), and cloned into the XbaI/EcoRIdigested vector pMORPH®X9_Fab_FH. Fabs expressed in these vectors carrytwo C-terminal tags (FLAG™ and 6×His, respectively) for detection andpurification.

D. Microexpression of HuCAL GOLD® Fab Antibodies in E. coli

Single colonies obtained after subcloning into pMORPH®X9_Fab_FH wereused to inoculate wells of a sterile 96-well microtiter plate containing100 μl 2×TY/Cm/1% Glu medium per well and grown overnight at 37° C. 5 μlof each TG-1 E. coli culture was transferred to a new sterile 96-wellmicrotiter plate containing 100 μl 2×TY/Cm/0.1% Glu medium per well.Microtiter plates were incubated at 30° C. shaking at 400 rpm on amicroplate shaker until the cultures were slightly turbid (˜2-4 hrs)with an OD_(600nm) of 0.5.

To these expression plates, 20 μl 2×YT/Cm/3 mM IPTG were added per well(end concentration 0.5 mM IPTG), sealed with a gas-permeable tape andincubated overnight at 30° C. shaking at 400 rpm.

Generation of Whole Cell Lysates (BEL Extracts)

To each well of the expression plates, 40 μl BEL buffer (2×BBS/EDTA:24.7 g/l boric acid, 18.7 g NaCl/l, 1.49 g EDTA/l, pH8) was addedcontaining 2.5 mg/ml lysozyme and incubated for 1 h at 22° C. on amicrotiter plate shaker (400 rpm). BEL extracts were used for bindinganalysis by ELISA or a BioVeris M-series® 384 analyzer (see Example 2).

E. Expression of HuCAL® GOLD Fab Antibodies in E. coli and Purification

Expression of Fab fragments encoded by pMORPH®X9_Fab_FH in TG-1 cellswas carried out in shaker flask cultures with 1 l of 2×YT mediumsupplemented with 34 μg/ml chloramphenicol. After induction with 0.5 mMIPTG, cells were grown at 22° C. for 16 h. Whole cell extracts of cellpellets were prepared by French Press and Fab fragments isolated bynickel/NTA chromatography (Qiagen, Hilden, Germany). Concentrations weredetermined by UV-spectrophotometry (Krebs et al., 2001).

Example 2: Identification of hGM-CSF Specific Antibodies

BEL extracts of individual E. coli clones selected by the abovementioned panning strategies were analyzed by ELISA or BioVeris(BioVeris M-series® 384 analyzer) in order to identify clones encodinghGM-CSF specific Fabs.

A. Enzyme Linked Immunosorbent Assay (ELISA) Techniques

Human recombinant biotinylated GM-CSF (R&D Systems) was coated at 1.5μg/ml in PBS onto Neutravidin microtiter plates for 2 h at RT.

After coating of antigen the wells were blocked with PBS/0.05% Tween(PBS-T) with 1% BSA for 1 h at RT. After washing of the wells with PBS-TBEL-extract, purified HuCAL® Fab or control IgGs were diluted in PBS,added to the wells and incubated for 1 h at RT. For detection of theprimary antibodies, the following secondary antibodies were applied:alkaline phospatase (AP)-conjugated AffiniPure F(ab′)₂ fragment, goatanti-human, -anti-mouse or -anti-rat IgG (Jackson Immuno Research). Forthe detection of AP-conjugates fluorogenic substrates like AttoPhos(Roche) were used according to the instructions by the manufacturer.Between all incubation steps, the wells of the microtiter plate werewashed with PBS-T three times and three times after the final incubationwith secondary antibody. Fluorescence was measured in a TECANSpectrafluor plate reader.

B. Electrochemiluminescene (BioVeris) Based Binding Analysis forDetection of GM-CSF Binding Fab in Lysates

Alternative to the ELISA experiments for the detection of GM-CSF bindingFab antibodies in E. coli lysates (BEL extracts), binding was analyzedin BioVeris M-SERIES® 384 AnalyzerBioVeris, Europe, Witney, Oxforfshire,UK).

To this end BEL extract was diluted at least 1:50 and maximally 1:1000in assay buffer (PBS/0.05% Tween20/0.5% BSA) for use in BioVerisscreening. Biotinylated GM-CSF (R&D Systems) was coupled to streptavidincoated paramagnetic beads, Dynabeads (Dynal), at a concentration of 0.1μg/ml. Per well of a 96 or 384 well plate 25 or 15 μl of a 1:25 dilutionof the Dynabead-stock solution was used. Beads were washed three timeswith assay buffer before adding biotinylated GM-CSF for 30 min at RT ona shaker. Beads were then washed three times with assay buffer andfinally resuspended in fresh assay buffer. Anti-human (Fab)′₂ (Dianova)was ruthenium labelled using the BV-tag™ (BioVeris Europe, Witney,Oxfordshire, UK). This secondary antibody was added to the GM-CSFcoupled beads at a concentration of 6 μg/ml immediately before use. 100μl or 60 μl of diluted BEL extract (see above) of E. coli expressioncultures containing Fab antibodies was filled into wells of a 96 or 384well plate and, respectively, 25 or 15 μl of the GM-CSF coupled beadsplus anti-Fab-BV-tag™ secondary antibody mix was added to each well andincubated for 2 h at RT on a plate shaking device. The plates wereanalyzed in a BioVeris M-SERIES® 384 Analyzer.

After sequence analysis seventy-four (74) unique clones were identifiedthat showed sufficiently strong binding (signal:noise ratio greater than10:1 in ELISA or 50:1 in BioVeris). These clones were expressed,purified and were tested in functional assays.

C. Determination of the Molecular Specificity and SpeciesCrossreactivity of Selected Anti-hGM-CSF Fabs.

Crossreactivity of the anti-hGM-CSF antibodies was determined to thefollowing analytes: rat and mouse GM-CSF, human IL-3, human IL-4, humanIL-5, human IL-13, human M-CSF (all from Peprotech, London, UK). Thiswas performed in a capture set-up by surface plasmon resonance (Biacore3000, Uppsala, Sweden). CM5 chips (Biacore, Sweden) were coated with5000-6000RU anti-F(ab)₂ (Dianova, Affinipure F(ab)₂ Fragment GoatAnti-Human IgG, F(ab)₂ Fragment specific); 80 μg/ml 10 mM acetatebuffer, pH4 on all 4 flow cells, using standard EDC-NHS amine couplingchemistry. On the flow cells 2-4 specific GM-CSF Fabs (20 μl of 500 nMFab at a flowrate of 5 μl/ml, 300-400RU) were captured. After capturingof the specific Fab, buffer was injected, to determine the dissociationof anti-Fab/Fab interaction. In a following cycle, the analyte growthfactor was injected (20 μl, flow rate 20 μl/min) at a concentrationrange between 15 and 2000 nM for the determination of the specificsignal. Afterwards the achieved buffer sensogram was manually subtractedfrom the specific one. After each cycle, the flow cells were regeneratedwith 100 mM HCl (5 μl). Seven HuCAL® anti-hGM-CSF antibodies includingMOR03684 and MOR03682 were tested and were specific for human GM-CSF anddid not bind to any of the other cytokines or mouse or rat GM-CSF. Incontrast Fab MOR03929 showed significant cross reactivity to rat GM-CSF.

Example 3: Identification of Anti-Human GM-CSF Fab Candidates thatInhibit the Interaction Between GM-CSF and the GM-CSF Receptor Alpha

74 different hGM-CSF specific antibodies which were selected from theHuCAL GOLD® library were tested for the potency to inhibit theinteraction between hGM-CSF and its receptor. The interaction was testedin two ways, (i) one being a proliferation assay using the GM-CSFdependent TF-1 cell line (Kitamura et al., 1989) and (ii) the otherbeing a FACS analysis with a recombinant CHO cell line expressing thealpha chain of the GM-CSF receptor. In the TF-1 proliferation assay, theability of the anti-GM-CSF antibodies to block the interaction of GM-CSFwith the endogenous GM-CSF receptor consisting of the alpha and betachain was analyzed leading to reduction in cell proliferation. In theFACS assay the specific inhibition of the interaction between GM-CSF andthe alpha chain of the GM-CSF receptor was determined.

A. Cloning and Expression of Macaca Mulatta and Human GM-CSF

Macaca mulatta GM-CSF full-length cDNA (Gen Bank accession no.:AY007376) was synthesized by gene synthesis (geneART GmbH, Regensburg,Germany) and cloned into the pCR-Script-Amp vector (Stratagene, LaJolla,Calif., USA). Subsequently the cDNA was cloned into the eukaryoticexpression vector pcDNA3.1 (+) (Invitrogen, Paisley, UK) yieldingpcDNA-macGM-CSF. The cDNA of human GM-CSF (Genbank accession numberNP_000749) was cloned by RT-PCR technique from RNA isolated from 1×10e7TF-1 cells using the RNeasy kit from Qiagen (Hilden, Germany). Reversetranscription was performed with the Superscriptll kit using randomhexamers (Gibco) followed amplification of the GM-CSF cDNA by PCR. Theobtained PCR-product was cloned into expression vector pcDNA3.1(+)yielding pcDNA-huGM-CSF.

HEK293 cells were transiently transfected with these expression vectorsrespectively using lipofectamine (Stratagene, LaJolla, USA). The mediumcontaining the secreted recombinant macaca or human GM-CSF was harvested4 days after transfection.

B. Inhibition of GM-CSF Dependent Proliferation of TF-1 Cells byAnti-hGM-CSF Fabs Using Human or Macaca GM-CSF

TF-1 (Kitamura et al., 1989) cells were grown according to theprovider's protocol (DSMZ, Braunschweig, Germany; DSMZ No. ACC 334).TF-1 cells were washed twice with RPM11640 medium (10% FCS) and thenseeded at a concentration of 2×10⁵ cells/ml in 50 μl per well of a flatbottom 96 well cell culture dish. Human recombinant GM-CSF (“Leucomax”,ESSEX Pharma, Munchen) at 0.5 ng/ml and HuCAL® Fab antibodies (200ng/ml-200 μg/ml diluted in RPM11640 medium, 10% FCS) were mixed for 30min and 50 μl of the mix was added to the TF-1 cells, so that the finalconcentration of GM-CSF was 0.25 ng/ml. Maximal cell proliferation (0%inhibition) was measured incubating TF-1 cells at a final GM-CSF ofconcentration of 0.25 ng/ml, without the addition of antibody. 100%inhibition of TF-1 proliferation was measured by omitting GM-CSF fromthe assay and keeping the cells in RPM11640 medium (10% FCS) only. TF1cells were then incubated for 72 hours at 37° C. with 5% CO₂ in ahumidified chamber. Cell vitality was measured by adding MTT or XTTreagent (Roche, Mannheim, Germany) according to the manufacturer'srecommendation. Overall 19 Fab were identified that showed significantinhibition of TF-1 proliferation. The binders MOR03682, MOR03684 andMOR03929 showed consistent inhibition of TF1 cell proliferation ofgreater than 50% at a concentration of 2 μM. The inhibitory activity ofthese non-optimized Fabs was not strong enough to determine an IC₅₀dose, because full inhibition could not be achieved. In comparison,monoclonal antibodies BVD2-21C11 (BD Biosciences Pharmingen; Cat#554503)and MAB215 (R&D Systems; Cat#MAB215) were able to fully inhibit TF-1proliferation. Additionally, binding of MOR03682 and MOR03684 to nativehuman GM-CSF was tested in the TF-1 proliferation assay. Instead ofadding purified human recombinant GM-CSF to the TF-1 cells a supernatantof 5637 cells (DSMZ No. ACC 35) that secrete native human GM-CSF intothe medium was used. From a dose response curve comparing the effect ofrecombinant human GM-CSF with different dilutions of the 5637supernatant it was determined that the medium contained ˜5 ng/ml ofnative human GM-CSF. By preincubation of the 5637 supernatant withanti-human GM-CSF Fab MOR03682 or MOR03684 the binding of native humanGM-CSF to the TF-1 cells was blocked so that the viability of cells wasreduced comparably to the experiment with recombinant human GM-CSF.MOR03684 and MOR03682 thus binds to native human GM-CSF. Fab MOR03929was not tested in this assay.

Additionally, the cross reactivity to macaca GM-CSF was tested in theTF-1 proliferation assay. Instead of adding purified human GM-CSF to theTF-1 cells a supernatant of transfected HEK293 cells that secreterecombinant macaca GM-CSF into the medium was used.

TF-1 cells proliferated in the presence of macaca GM-CSF containingsupernatant but not in the presence of supernatant from untransfectedHEK293 cells. From a dose response curve comparing the effect ofrecombinant human GM-CSF with different dilutions of the HEK-293 mediumit was determined that the medium of the transfected cells contained ˜2μg/ml macaca GM-CSF. By preincubation of the macaca GM-CSF supernatantwith anti-human GM-CSF Fab MOR03682 or MOR03684 the binding of macacaGM-CSF to the TF-1 cells was blocked so that the viability of cells wassignificantly reduced. MOR03682 and MOR03684_thus are cross-reactivewith macaca GM-CSF. Fab MOR03929 was not tested in this assay.

C. Blocking of GM-CSF Binding to the GM-CSF Receptor Alpha byAnti-hGM-CSF Fabs

In order to test binding of GM-CSF to a cell surface expressed GM-CSFreceptor alpha chain the cDNA was cloned into an expression vector andstably transfected into CHO-K1 cells (DSMZ ACC 110).

Cloning of a Stable CHO-K1 Cell Line Expressing the Alpha Chain of theGM-CSF Receptor

The cDNA of the human GM-CSF receptor alpha chain (Genbank accessionnumber M64445) was cloned by RT-PCR technique from RNA isolated from1×10e7 TF-1 cells using the RNeasy kit from Qiagen (Hilden, Germany).Reverse transcription was performed with the Superscriptll kit usingrandom hexamers (Gibco). The GM-CSF-receptor alpha chain cDNA was thenamplified using the following primers:

(SEQ ID NO: 64) 5′: N-GCRa-plusSS:TTCTCTGGATCCGCCACCATGCTTCTCCTGGTGACAAGCC and (SEQ ID NO: 65)3′: C-flGCRa: ACCCTCCAATTGTCAGGTAATTTCCTTCACGGTC.

The PCR reaction yielded a product of ˜1250 bp which was digested withEcoRI and BamHI (New England BioLabs). The expression vector pcDNA3.1(+)(Invitrogen, Paisley, UK) was digested with the same enzymes. Afterpurification of the digested vector and PCR product, the fragments wereligated and transformed by electroporation into E. coli DH10B cells.Correct clones were identified after preparation of plasmid DNA andsequencing. Correct clones (pcDNA3.1(+)-GM-CSFRalpha) contained the fulllength human GM-CSF receptor alpha cDNA. CHO-K1 cells were grownaccording to the provider's protocol (DSZM, Braunschweig, Germany; DSMZNo. ACC 110). For transfection cells were grown to 80% confluence in a6-well plate and incubated with 5 μg DNA of pcDNA3.1(+)-GM-CSFRalphamixed with 10 μl of the Lipofectamine 2000 reagent (Invitrogen). After48 h cells were fed with 1 mg/ml G418 (Gibco) and after another 24 hmedium was replaced with such containing 2 mg/ml G418. After two weekssingle cells were seeded into wells of a 96-well culture dish. Singleclones were grown up and 5×10⁵ cells of each clone were tested forGM-CSFR-alpha expression by FACS analysis using murine IgG MAB1006(Chemicon International, Temecula, Calif.) as primary antibody at aconcentration of 1 μg/ml and (R-PE-AffiniPure (Fab′)₂Goat-anti-mouse-IgG (Dianova) as secondary antibody at a 1:200 dilution.Primary and secondary antibodies were incubated with the cells for 1 hsequentially, while cells were washed in FACS buffer (PBS, 3% FCS)between these steps. Fluorescence of stained cells was quantified in theFL2 channel using a FACSCalibur system (Becton Dickinson). Among theclones analyzed clone CHO-GMRa#11 showed the highest median fluorescentintensity. A median fluorescence value (MFL value) of 157 was determinedfor CHO-GMRa#11 (FIG. 6)

FACS Analysis of GM-CSF Binding to the GM-CSF Receptor Alpha Expressedon CHO-GMRa#11:

Prior to adding to cells, antibodies at increasing concentrations (0.1to 100 μg/ml) were co-incubated with biotinylated GM-CSF (0.5 μg/ml) inFACS buffer (PBS/3% FBS/NaN₃0.05%) for 30 min at RT.

All stainings were performed in round bottom 96-well culture plates(Nalge Nunc) with 1-5×10⁵ cells per well. 2×10E5 CHO-GMRa#11 cells weretaken up in 50 μl of the antibody/GM-CSF containing FACS buffer andincubated at 4° C. for 1 h. Cells were then washed once with 150 μl FACSbuffer/well and taken up in 100 □μl phycoerythrin-labeled streptavidin(BD Biosciences Pharmingen) which has been diluted 1:400 in FACS buffer.After 1 h incubation at 4° C. cells were washed twice with FACS buffer,resuspended in 100 □μl of FACS buffer and binding of biotinylated GM-CSFwas measured via FL2 fluorescence intensity of cells in FACScalibur(Becton Dickinson). IC₅₀ values were determined from the dose responsecurves obtained using GraphPad Prism v3.03 software applying anon-linear regression curve fit. Fab antibodies MOR03682, MOR03684 andMOR03929 showed significant inhibition of GM-CSF binding to the cellsurface expressed GM-CSF receptor alpha.

Example 4: Affinity Maturation of Selected Fab by Stepwise Exchange ofCDR Cassettes

A. Generation of Affinity Maturation Fab Libraries and Pannings

In order to increase the affinity and inhibitory activity of theanti-GM-CSF Fab fragments clones MOR03682, MOR03684 and MOR03929 weresubjected to affinity maturation. In this regard, CDR regions wereoptimized by cassette mutagenesis using trinucleotide directedmutagenesis (Virnekäs et al., 1994; Nagy et al., 2002). Sequenceanalysis revealed no sequence homology between the CDRs of the threeparental clones analyzed. Table 2a and 2b provide the six CDR peptidesequences for the parental clones MOR03682, MOR03684 and MOR03929.

The following briefly describes the protocol used for Fab optimization.Fab fragments from expression vector pMORPH®X9Fab_FH were cloned into aphagemid vector (U.S. Pat. No. 6,753,136). Then, two differentstrategies were applied to optimize the affinity and efficacy of theparental Fabs.

First, one phage antibody Fab library was generated where the L-CDR3 ofeach parental was replaced by a repertoire of individual lambda lightchain CDR3 sequences. In a second library the H-CDR2 region was replacedby a repertoire of individual heavy chain CDR2 sequences.

Affinity maturation libraries were generated by transforming thediversified clones into E. coli TOP10F′ (Invitrogen). Phages wereprepared as described in Example 1A. Both L-CDR3 libraries of MOR03684and MOR03682 were pooled and both H-CDR2 libraries derived from MOR03684and MOR03682 were pooled, while the L-CDR3 and H-CDR2 libraries derivedfrom MOR03929 were kept separately during the selection procedure.

Pannings were performed on biotinylated GM-CSF in solution for threerounds essentially as described in Example 1B and applying morestringent selection conditions.

B. Electrochemiluminescene (BioVeris) Based Binding Analysis forDetection of Improved GM-CSF Binding Fab in Lysates

For the detection of GM-CSF binding Fab antibodies in E. coli lysates(BEL extracts), binding was analyzed in the BioVeris M-384 SERIES®Workstation (BioVeris Europe, Witney, Oxforfshire, UK) essentially asdescribed in Example 2B.

Fabs with the highest ECL values were purified and subjected to affinitymeasurement by solution equilibrium titration (SET; Haenel et al, 2005)and surface plasmon resonance (Biacore) (see Example 4D)

C. X-Cloning of Improved VL (L-CDR3) with Improved VH (H-CDR2)

For a further improvement of affinity the independently optimized H-CDR2and L-CDR3 from matured Fabs which were derived from the same parentalclone were combined, because there was a high probability that thiscombination would lead to a further gain of affinity (Yang et al., 1995;Schier et al., 1996; Chen et al., 1999). This procedure, calledX-cloning, was applied for binders that were derived from the parentalclone MOR03929 as Fabs with improved affinities were identified fromboth the H-CDR2 and the L-CDR3 library. This was accomplished bytransferring whole light chains (XbaI/SphI fragment) from theL-CDR3-optimized donor clone to the H-CDR2-optimized acceptor clone.

TABLE 3 X-cloning combinations Affinity Improved Fab Parental VH donorVL donor after X-cloning MOR03929 MOR04287 × MOR04302 MOR04350 MOR04290MOR04354 MOR04252 MOR04357 MOR03682 MOR04283 × MOR04297 MOR04342

For the resulting 4 Fabs the VL and VH was sequenced to confirm transferof the correct VL to the respective H-CDR2 improved vector backbone.Table 2a and 2b show the VH and VL protein sequences of all derivativesof MOR03929 and 3682, which are listed in table 3.

D. Determination of Picomolar Affinities Using Solution EquilibriumTitration (SET) and Surface Plasmon Resonance (Biacore)

For K_(D) determination, monomer fractions (at least 90% monomercontent, analyzed by analytical SEC; Superdex75, Amersham Pharmacia) ofFab were used. Electrochemiluminescence (ECL) based affinitydetermination in solution and data evaluation were basically performedas described by Haenel et al., 2005: A constant amount of Fab wasequilibrated with different concentrations (serial 5^(n) dilutions) ofhuman GM-CSF (Leucomax) in solution. Biotinylated human GM-CSF (R&DSystems) coupled to paramagnetic beads (M-280 Streptavidin, Dynal) andBV-tag™ (BioVeris Europe, Witney, Oxforfshire, UK) labeled anti-human(Fab)′₂ (Dianova) was added and incubated for 15-30 min. Subsequently,the concentration of unbound Fab was quantified via ECL detection usinga M-SERIES® 384 analyzer (BioVeris Europe).

In accordance with Friguet et al., 1985, care was taken to avoidsignificant equilibrium shift to solid phase during detection.

Using the assay conditions described above affinities for the Fabs weredetermined, which are shown in table 4.

Additionally kinetic SPR analysis was performed on an F1 chip (Biacore,Sweden) which was coated with a density of ˜100 RU recombinant humanGM-CSF (Peprotech) in 10 mM Na-acetate pH 4.5 using standard EDC-NHSamine coupling chemistry. A respective amount of HSA was immobilized onthe reference flow cell. PBS (136 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄,1.76 mM KH₂PO₄ pH 7.4)+0.005% Tween 20 was used as running buffer. Fabwas applied in concentration series of 6.3-200 nM at a flow rate of 20μl/min. Association phase was set to 60 s and dissociation phase to 120s (parental) or up to 600 s (affinity optimized). In order to monitordissociation phase over a longer period, the following conditions,basically according to Drake et al., (2004) were used: Fab was appliedin a single concentration of 200 nM; flow rate was set to 100 μl/min anddissociation phase to 6000-18.000 s. On the basis of the off-ratesdetermined under these assay conditions affinities for the Fabs wereestimated, which are shown in table 4.

TABLE 4 Affinities of anti-hGM-CSF Fabs determined by Biacore andsolution equilibrium titration (SET) Biacore SET MOR0 K_(D) (pM) K_(D)(pM) 3684 6420 16000 4251 70 7.4 3929 4260 2000 4302 174 63.5 4287 nd17.9 4252 55 6 4290 122 11.1 4350 19 1.1 4354 21 2.8 4357 7 0.4 3682 nd11406 4283 nd 113 4297 nd 49.2 4342 nd 4.9

E. Determination of Affinities to Rat GM-CSF Using Solution EquilibriumTitration (SET)

Affinity determination to rat GM-CSF was done essentially as describedin Example 4D using rat-GM-CSF (Peprotech) as analyte in solutioninstead of human GM-CSF. Affinities were calculated according to Haenelet al (2005). In this assay affinity of Fab MOR04357 to rat GM-CSF wasdetermined to be K_(D)=1.0 nM.

Example 5: Characterization of Optimized Anti-Human GM-CSF Fabs thatInhibit the Interaction Between GM-CSF and the GM-CSF Receptor AlphaChain

A. GM-CSF Receptor Alpha Binding Assay

The GM-CSF receptor binding assay was performed as described above(Example 3C) using 0.5 μg/ml (35 nM) of biotinylated GM-CSF. Maximalbinding of GM-CSF to CHO-GMRa#11 cells (0% inhibition) was measured byincubating cells at a final GM-CSF concentration of 0.5 μg/ml ofbiotinylated GM-CSF, without the addition of antibody. 100% inhibitionof GM-CSF binding was measured by omitting GM-CSF from the assay. IC₅₀values were determined from the dose response curves obtained usingGraphPad Prism v3.03 software applying a non-linear regression curvefit. Fabs with improved affinities, the parental Fabs and monoclonalreference IgGs were analyzed. Table 5 summarizes the IC50 valuesobtained in these assays. The % inhibition achieved at an antibodyconcentration of 5 μg/ml is also given in table 5.

TABLE 5 IC₅₀ values of anti-hGM-CSF Fabs in receptor inhibition assayMOR0# 4251 4357 4354 4350 4252 4287 4290 4302 3684 3929 Mab215 21C11IC₅₀ (nM) 53 26 26 24 26 25 27 24 >400 35 no fit* 9 % inhibition ~100%~75% ~75% ~75% ~75% ~75% ~75% ~75% ~25% ~60% ~50% ~100% at 5 μg/mlantibody *no sigmoidal dose response curve could be fitted in this case

This assay qualitatively showed that the Fabs obtained from affinitymaturation and X-cloning prevent GM-CSF from binding to the GM-CSFreceptor alpha chain and therefore retained the blocking mechanism oftheir parental Fabs. The assay needed to be performed with aconcentration of 35 nM (0.5 μg/ml) biotinylated GM-CSF in order toobtain a significant signal in FACS. Therefore 17.5 nM Fab (or 8.75 nMIgG) is theoretically needed to block 50% of the GM-CSF, thus setting alimit for determination of IC₅₀ values.

B. Inhibition of GM-CSF Dependent Proliferation of TF-1 by Anti-hGM-CSFFabs Using Human GM-CSF

TF-1 proliferation assay was performed as described in Example 3B. Fabwith improved affinities and the parental Fabs as well as monoclonalreference IgGs were analyzed. IC₅₀ values were determined from the doseresponse curves obtained using GraphPad Prism v3.03 software applying anon-linear regression curve fit. Table 6 summarizes the IC₅₀ valuesobtained in these assays.

TABLE 6 IC₅₀ values of anti-hGM-CSF Fabs and control IgGs in TF-1proliferation assay BVD2- MOR0# 4251 4357 4354 4350 4252 4287 4290 43023684 3929 Mab215 21C11 IC₅₀ (pM) 463 90 56 82 2010 3382 69610678 >200000 >200000 4315 6560 IC₅₀ 9.3 47.9 77.1 52.6 2.1 1.3 6.2 — —— — — x-fold improved compared to Mab215 IC₅₀ 14.2 72.9 117.1 80.0 3.21.9 9.4 — — — 1.5 — x-fold improved compared to BVD2- 21C11

In another set of experiments IC₅₀ values in the TF-1 proliferationassay were determined for the parental Fab MOR03682, its affinitymatured derivatives MOR04283, MOR04297 and the x-cloned variantMOR04342. Table 7 summarizes the IC₅₀ values obtained in these assays.

TABLE 7 IC₅₀ values of anti-hGM-CSF Fabs in TF-1 proliferation assayMOR0# 4342 4283 4297 3682 IC₅₀ (pM) 80 17293 13975 >200000

These experiments demonstrated the large improvements achieved in IC₅₀values after affinity maturation and X-cloning. For example, MOR04357,MOR04350, MOR04354 show >2000 fold improved IC₅₀ values compared totheir parental MOR03929 and exceed the potency of BVD2-21011 and Mab215

Example 6: Conversion of MOR04357 to Human IgG1 Format

A. Gene Optimization of Fab DNA Sequences for Expression in MammalianExpression Systems.

To optimize DNA of the VH and VL of MOR04357 for mammalian geneexpression (e.g. changing codon usage, GC content, etc.) GeneOptimizer™software from Geneart (Regensburg, Germany) was utilized to define suchoptimized VH and VL DNA sequences, which were gene synthesized atGeneart (Regensburg, Germany) and cloned into pPCR-Script vectorsyielding 055906pPCR-Script and 055907pPCR-Script. SEQ ID NO: 48 showsthe respective VH sequence, while SEQ ID NO: 57 shows the respective VLsequence.

B. Cloning of Fab MOR04357 into Human IgG1 Format and IgG1 Expression

In order to express full length immunoglobulin (Ig), variable domainfragments of the gene optimized heavy (VH) and light chains (VL) weresubcloned from the pPCR-Script vectors (Example 5a) into the thepMORPH®2_h_lg vector series for human IgG1. Codon-optimized VH ofMOR04357 was isolated from 055906pPCR-Script via NheI/BlpI digestion andinserted into pMorph2_h_IgG1f master vector cut with the samerestriction enzymes. This vector already contained a human gamma 1constant region. The resulting expression plasmid was termedpMorph2_h_IgG1f_MOR04357_co. Codon-optimized VL of MOR04357 was isolatedfrom 055907pPCR-Script via NheI/HpaI digestion and inserted intopMorph2_h_Iglambda2 master vector cut with the same restriction enzymes.This vector already contained a human lambda constant region. Theresulting expression plasmid was termed pM2_h_Iglambda2_MOR04357_co.

C. Transient Expression and Purification of Human IgG

Eukaryotic HKB11 cells were transfected with an equimolar amount of IgGheavy and light chain expression vector DNA. Cell culture supernatantwas harvested from 3 to 7 days post transfection. After adjusting the pHof the supernatant to 8.0 and sterile filtration, the solution wassubjected to standard protein A affinity chromatography (rProteinA FF orMabSelect SURE, GE Healthcare). Buffer exchange was performed to 1×Dulbcecco's PBS (pH 7.2, Invitrogen) and samples were sterile filtered(0.2 μm). MOR04357 IgG1 was dialysed against 1× Dulbcecco's PBS (pH 6.5,Invitrogen). Purity of IgG was analysed under denaturing, reducingconditions in SDS-PAGE or by using Agilent BioAnalyzer and in nativestate by SE-HPLC.

D. Determination of Picomolar Affinities Using Solution EquilibriumTitration (SET)

For K_(D) determination, monomer fractions (at least 90% monomercontent, analyzed by analytical SEC; Superdex75, Amersham Pharmacia) ofIgG1 were used. Electrochemiluminescence (ECL) based affinitydetermination in solution and data evaluation were basically performedas described by Haenel et al., 2005 and as described in Example 4B. TheK_(D) values for MOR04357 IgG1 against human recombinant GM-CSF wasdetermined to be 1.1 pM.

E. Determination of Affinities to Rat GM-CSF Using Solution EquilibriumTitration (SET)

Affinity determination to rat GM-CSF was done essentially as describedin Example 4D using rat-GM-CSF (Peprotech) as analyte in solutioninstead of human GM-CSF. Affinities were calculated according to Haenelet al (2005). The K_(D) value for the MOR04357 IgG1 against ratrecombinant GM-CSF was determined to be 130 pM.

Example 7: Characterization of MOR04357 IgG1 Derived from OptimizedAnti-Human GM-CSF Fabs

A. Inhibition of GM-CSF Dependent Proliferation of TF-1 by Anti-hGM-CSFIgGs Using Human and Rhesus GM-CSF

TF-1 proliferation assay was performed as described in Example 3B.MOR04357 was analyzed in IgG1 format and as control monoclonal referenceIgGs were analyzed. IC₅₀ values were determined from the dose responsecurves obtained using GraphPad Prism v3.03 software applying anon-linear regression curve fit. Table 8 summarizes the IC₅₀ valuesobtained in these assays. Three different variants of GM-CSF were usedin this assay: Firstly, recombinant human GM-CSF at a concentration of0.25 ng/ml, produced in E. coli, secondly, culture supernatant fromHEK293 which have been transiently transfected with pcDNA-huGM-CSF (seeExample 3A), containing recombinant human GM-CSF and thirdly, culturesupernatant from HEK293 cells which have been transiently transfectedwith pcDNA-macGM-CSF (see Example 3A), containing recombinant macacamulatta (rhesus) GM-CSF. For TF-1 proliferation assays the HEK293culture supernatants were used as a source of the respective GM-CSF insuch dilutions that TF-1 cells showed a similar proliferation ascompared to proliferation given at the defined concentration of 0.25ng/ml purified recombinant human GM-CSF produced in E. coli.

TABLE 8 IC₅₀ values of MOR04357 IgG and control IgGs in TF-1proliferation assay IC₅₀ (pM) human human macaca GM-CSF GM-CSF GM-CSF(E. coli) (HEK293) (HEK293) MOR04357 IgG1 48 11 15 21C11 1668 144 128Mab215 625 54 190

This experiment demonstrated the large improvements achieved in IC₅₀values after affinity maturation and X-cloning where preserved afterconversion from Fab to IgG1 format. IgG1 MOR04357 shows >2000 foldimproved IC₅₀ values compared to Fab MOR03929 and exceeds the potency ofBVD2-21C11 and Mab215.

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1.-31. (canceled)
 32. A GM-CSF binding protein comprising: (a) a VHregion comprising a sequence at least 80% identical to the sequence ofSEQ ID NO:20; or (b) a VL region comprising a sequence at least 80%identical to the sequence of SEQ ID NO:40.
 33. The GM-CSF bindingprotein of claim 32 comprising: (a) a VH region comprising a sequence atleast 80% identical to the sequence of SEQ ID NO:20; and (b) a VL regioncomprising a sequence at least 80% identical to the sequence of SEQ IDNO:40.
 34. The GM-CSF binding protein of claim 32 comprising: (a) a VHregion comprising a sequence at least 90% identical to the sequence ofSEQ ID NO:20; or (b) a VL region comprising a sequence at least 90%identical to the sequence of SEQ ID NO:40.
 35. The GM-CSF bindingprotein of claim 32 comprising: (a) a VH region comprising a sequence atleast 90% identical to the sequence of SEQ ID NO:20; and (b) a VL regioncomprising a sequence at least 90% identical to the sequence of SEQ IDNO:40.
 36. The GM-CSF binding protein of claim 32 comprising: (a) a VHregion comprising a sequence at least 95% identical to the sequence ofSEQ ID NO:20; or (b) a VL region comprising a sequence at least 95%identical to the sequence of SEQ ID NO:40.
 37. The GM-CSF bindingprotein of claim 32 comprising: (a) a VH region comprising a sequence atleast 95% identical to the sequence of SEQ ID NO:20; and (b) a VL regioncomprising a sequence at least 95% identical to the sequence of SEQ IDNO:40.
 38. The GM-CSF binding protein of claim 32, wherein the bindingprotein comprises a synthetic polypeptide, a humanised sequence, or achimeric sequence.
 39. The GM-CSF binding protein of claim 33, whereinthe binding protein comprises a synthetic polypeptide, a humanisedsequence, or a chimeric sequence.
 40. A nucleic acid sequence whichencodes the binding protein as claimed in claim
 32. 41. A nucleic acidsequence which encodes the binding protein as claimed in claim
 33. 42.An expression vector comprising the nucleic acid sequence as claimed inclaim
 40. 43. An expression vector comprising the nucleic acid sequenceas claimed in claim
 41. 44. A recombinant host cell comprising thenucleic acid sequence as claimed in claim
 40. 45. A recombinant hostcell comprising the nucleic acid sequence as claimed in claim
 41. 46. Arecombinant host cell comprising the nucleic acid sequence as claimed inclaim
 42. 47. A recombinant host cell comprising the nucleic acidsequence as claimed in claim
 43. 48. A method for the production of aGM-CSF binding protein, which method comprises culturing a host cell asdefined in claim 46 under conditions suitable for expression of saidnucleic acid sequence, whereby a polypeptide comprising the GM-CSFbinding protein is produced.
 49. A method for the production of a GM-CSFbinding protein, which method comprises culturing a host cell as definedin claim 47 under conditions suitable for expression of said nucleicacid sequence, whereby a polypeptide comprising the GM-CSF bindingprotein is produced.
 50. A pharmaceutical composition comprising thebinding protein as claimed in claim 32 and one or more pharmaceuticallyacceptable carriers, excipients or diluents.
 51. A pharmaceuticalcomposition comprising the binding protein as claimed in claim 33 andone or more pharmaceutically acceptable carriers, excipients ordiluents.
 52. A method for the treatment of rheumatoid arthritis in asubject in need thereof comprising administering to said subject atherapeutically effective amount of the binding protein as defined inclaim 32 to the subject.
 53. A method for the treatment of rheumatoidarthritis in a subject in need thereof comprising administering to saidsubject a therapeutically effective amount of the binding protein asdefined in claim 33 to the subject.